Tongmai Huazheng mixture attenuates adenomyosis by inducing ferroptosis through suppression of the JAK2/STAT3 signaling pathway

Background: Ferroptosis is a newly characterized iron-dependent form of regulated cell death. In adenomyosis (AM), ectopic endometrial lesions exhibit resistance to Ferroptosis, which contributes to disease progression and recurrence. Tongmai Huazheng mixture (TMHZ) is a hospital-formulated traditional Chinese medicine (TCM) compound that has demonstrated clinical efficacy in the management of AM. However, the precise mechanisms by which TMHZ regulates Ferroptosis in AM remain unclear.

Purpose: The aim of this study was to determine whether TMHZ exerts its anti-AM effects by inducing Ferroptosis through inhibition of the JAK2/STAT3 signaling pathway.

Methods: A multiomics strategy integrating UPLC-MS/MS, network pharmacology, and RNA Sequencing was employed to identify candidate targets and key regulatory pathways of TMHZ. In vitro, the effects of TMHZ-containing serum on proliferation, migration, and invasion of adenomyosis-derived cells (AMDCs) were evaluated using CCK-8, EdU, wound healing, and Transwell assays. Ferroptosis induction and oxidative stress levels were assessed using FerroOrange and CM-H₂DCFDA fluorescent probes, glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) assay kits, and transmission electron microscopy. Immunofluorescence and western blotting (WB) were performed to investigate the regulatory effects of TMHZ on the JAK2/STAT3 pathway and ferroptosis-related proteins. The JAK2 Inhibitor AG490 and agonist coumermycin A1 were used to validate the involvement of the pathway. In vivo, a tamoxifen-induced AM mouse model was established to assess the therapeutic efficacy and safety of TMHZ. Histopathological changes in the uterus were examined by hematoxylin-eosin (HE) staining, and serum levels of estradiol (E2) and progesterone (P4) were measured to verify model establishment. SOD and MDA levels and the results of immunohistochemical and WB analyses were used to further confirm the in vivo regulation of Ferroptosis and JAK2/STAT3 signaling by TMHZ. Additionally, HE staining of major organs (liver, spleen, and kidney) was performed to evaluate systemic safety.

Results: Bioinformatics analysis suggested that the therapeutic effects of TMHZ were closely associated with the regulation of the JAK2/STAT3 pathway and Ferroptosis. In vitro, TMHZ significantly inhibited the proliferation, migration, and invasion of AMDCs; increased intracellular Fe²⁺ and Reactive Oxygen Species (ROS) levels; reduced GSH and SOD activity; elevated the MDA content, and induced typical mitochondrial damage. TMHZ also downregulated the expressions of p-JAK2, p-STAT3, SLC7A11, and GPX4. Further rescue experiments using JAK2 inhibitors and agonists confirmed the key role of the JAK2/STAT3 pathway in this process. In vivo, TMHZ suppressed JAK2/STAT3 signaling to promote Ferroptosis, markedly alleviated the pathological invasion of ectopic endometrial glands into the myometrium, and demonstrated a favorable systemic safety profile.

Conclusion: TMHZ inhibits the JAK2/STAT3 pathway, thus inducing Ferroptosis in ectopic lesions. This ferroptotic response suppresses the proliferation, migration, and invasion of AMDCs, ultimately contributing to the attenuation of AM progression. These findings offer novel mechanistic insights and support TMHZ as a potential therapeutic strategy for AM within the framework of TCM.

Adenomyosis; Ferroptosis; Reactive oxygen species; Traditional chinese medicine; UPLC-MS/MS.